Location: Emerging Pests and Pathogens Research2012 Annual Report
1a. Objectives (from AD-416):
1: Describe key bacterial pathways involved in disease establishment and progression. 1A: Characterize the role of ncRNAs and the RNA binding proteins Crc and Hfq in pathogenesis. 1B: Identify ECF sigma factor regulons and determine their role(s) in plant interactions. 2: Identify bacterial transcriptional responses to plant signals and defense systems in planta. 2A: In vitro, examine bacterial transcriptional responses to conditions thought to be relevant in planta. 2B: Examine bacterial transcription during plant infection. 2C: Evaluate each gene in P. syringae for its contribution to fitness in plants. 2D: Develop systems models of pathogen-plant interactions.
1b. Approach (from AD-416):
Bacterial plant pathogens are responsible for major losses in nearly all crops. Attempts to develop resistance in host plant species have been hindered by a lack of understanding of the complex network of plant-microbe interactions. A central problem is that the pathways used by bacteria to sense and respond to the environment inside the plant are largely unknown. Building upon our previous work in Pseudomonas syringae pv. tomato DC3000, we will address this problem by analyzing non-coding RNAs and ECF sigma factors, two classes of regulatory factors that are known linchpins of gene regulation. We will use established deep sequencing methods, such as ChIP-Seq, RNA-Seq and RNA 5'-end capture, and novel methods, such as genomic footprinting and in planta RNA-Seq, to monitor bacterial gene expression as it occurs during infection. The synthesis of the data sets from these experiments will reveal many key regulatory pathways involved in pathogenesis and virulence.
3. Progress Report:
In February 2012, the Unit finished work on the previous project (1907-21000-027-00D) and started work on this current project. Activities during this reporting period have focused on bringing the old project to conclusion and laying the foundation for the new project. The activities related to the old project are reported elsewhere in the Annual Report for (1907-21000-027-00D). Activities related to Objective 1 of the new project included further work on identifying and characterizing small RNAs (sRNAs) and identifying the regulon of DC3000’s iron-starvation (IS), extracytoplasmic function (ECF) sigma factors. For sRNAs, mutants were developed that produced Flag-tagged versions of the sRNA chaperone proteins, Hfq and Crc. Assays were performed to validate these constructs and protein-RNA complexes were isolated. The next step is to perform high-throughput sequencing of the RNA isolated from these complexes (RIP-Seq). For IS ECF sigma factors, a manuscript describing the regulon of PSPTO_1203 was published. Also, we identified the regulon of PSPTO_0444, PSPTO_1209 and PSPTO_1286. Although the regulons of the IS ECF sigma factors of DC3000 have been catalogued, which was one of the objectives of the previous project, many questions remain about what extracellular signals induce these regulatory systems. Activities related to Objective 2 included using high-throughput sequencing to simultaneously screen an entire transposon insertion library (Tn-Seq). The Tn-Seq protocol was designed and a pilot experiment was performed. This experiment demonstrated that the insertion library was successfully constructed, but improvements in the method for extracting transposon-genomes junctions are needed for this method to simultaneously screen all of the genes of the organism under a variety of environmental conditions. Also, during this reporting period, we have made substantial progress towards our milestones of integrating data sets and identifying models for DC3000 metabolic and plant immune signaling networks. Initial efforts have focused primarily on assembling data from our collaborators and from the literature that measures quantitative changes to DC3000 growth rates in planta either in DC3000 effector mutants or plant defense signaling mutants, and building models of those systems through the use of machine learning and network analysis techniques. Two Cornell graduate students have participated in this effort during rotation projects, and we continue to develop working relationships with our collaborators in Cornell Plant Pathology & Plant Microbe Biology and the Boyce Thompson Institute.
1. High-throughput capture and mapping of start of bacterial RNA molecules. Identifying the many interlocking signaling and regulation systems that pathogenic bacteria use to control the expression of their genes is critical to understanding these organisms and developing countermeasures. In order to identify the loci of their regulation activity, ARS researchers at Ithaca, New York developed a novel method for capturing and mapping the start of bacterial RNA molecules. By locating transcription start sites, it is possible to analyze the genomic neighborhood for regulatory elements that are active under specific environmental conditions. Using this method, thousands of transcription starts were readily mapped, which enabled hundreds of regulatory elements to be identified and assigned to specific genes. This method can be used in a wide range of bacteria, and is likely to enable other scientists to unravel the regulation networks of other agriculturally important organisms.